Stem cell replacement therapies are a promising, curative alternative to the long-term management approaches associated with locomotor deficits from trauma or neurodegeneration. However, while cell therapies for spinal cord regeneration are promising, studies to date have suffered from poor neuronal integration and/or variable functional outcomes. One reason for this may be regional phenotype mismatch. Studies in the brain have highlighted the importance of regional specification of human pluripotent stem cell (hPSC)-derived neural progenitors to alleviate Parkinson's, Huntington's, and Epilepsy symptoms in rodent models. Comparable studies in the spinal cord have been hindered by a limited capacity to control the regional phenotype of hPSC-derived spinal populations. The fundamental hypothesis of this proposal is that genetic specification of hPSC-derived neuronal transplants to discrete spinal cord regions significantly affects engraftment efficacy and subsequently patients' functional recovery. This work focuses on motor neurons (MNs), which are specifically targeted in a number of neurodegenerative diseases and are damaged following spinal cord injury. During neural tube development, colinear HOX expression results in spatial patterning of neuronal phenotypes along the R/C axis of the spinal cord. The Ashton lab has established protocols recapitulate this Hox progression in hPSCs, generating neural stem cells with discrete Hox profiles. When combined with morphogens for ventral patterning, this protocol enables the derivation of a full rostrocaudal spectrum of progenitor MNs (pMNs) and MNs that can serve as region-specific populations for transplantation. Aim 1 focuses on the generation and characterization of these regionalized MN cultures representative of high cervical, mid cervical, brachial, thoracic, lumbar, and sacral anatomical segments. In addition to characterization by molecular and functional assays, single cell RNA-seq will be performed to determine columnar and motor pool identities within regionalized MN populations prior to transplantation. Aims 2 and 3 test the hypothesis that regionalized hPSC-derived pMNs differentially integrate into host circuits and selectively enhance functional recovery in vivo. Aim 2 examines whether pMNs preferentially engraft into their region-matched spinal cord segment and selectively project axons onto coordinate musculature in a developmental chick model. Aim 3 seeks to determine whether regionalized pMNs contribute to functional recovery following transplantation into an adult rat that has been selectively ablated of phrenic MNs. The expectation is that behavioral gains are mitigated upon transplant silencing. Together, these aims establish clinically relevant MN populations for transplantation, advance a mechanistic understanding of human MN diversification and establish the role of regional specificity on neuronal integration into the central nervous system. The findings can guide future clinical interventions and are translatable to other spinal cells, including interneurons and glia.